Structural insights into plant phytochrome A as a highly sensitized photoreceptor

Dear Editor, Phytochromes (phys), ﬁ rst discovered in plants, are red and far-red photoreceptors that are also widely found in bacteria and fungi later. 1,2 Plant phys utilize the linear tetrapyrrole chromophore called photochromobilin (P Φ B). Phys reversibly toggle between the red light-absorbing Pr and the far-red light-absorbing Pfr conformers by photoconversion, with absorption peaks at ~667 nm and ~730 nm, respectively. The Pfr conformer can be converted to Pr in darkness, a process called dark reversion or thermoreversion. 2,3 In plants, Pr conformers reside in the cytoplasm; and upon photoconversion, Pfr conformers are translocated into the nucleus, resulting in a plethora of physiological and developmental changes throughout the plant life cycle. Thehigher plant Arabidopsis thaliana encodes ﬁ ve phy genes, designated phyA – E . 4 PhyB is the major red light receptor mediating the classical red/far-red light reversible low ﬂ uence response (LFR) or red light high irradiance response (R-HIR). PhyC – E mostly perform a complementary function to that of phyB in adult plants. 2,3 PhyA is distinct from other phys and is responsible for the very low ﬂ uence response (VLFR) under a broad spectrum of light and for the far-red light high irradiance response (FR-HIR). 2,3 Thus, phyA has higher photosensitivity than phyB and is intrinsically more sensitive to light. 5,6 In higher plants, phys share a conserved domain structure consisting of an N-terminal photosensory module (PSM) followed by two tandem Period/ARNT/Singleminded (PAS) domains, and a C-terminal

The integrity of the final phyA preparations was confirmed by UV280 absorption spectra, and by SDS-PAGE followed by staining of the gels either for protein with Coomassie Brilliant Blue or for PΦB by zinc-induced fluorescence under UV light.

UV-Vis Spectroscopy and kinetic analyses
Absorption spectra of Pr-to-Pfr photoconversion in Fig. 1l and Fig. S1 were measured by UV-1900i spectrophotometer (Shimadzu) at 25 °C in the dark.To maintain the consistency of experimental results, the cuvette and sample volumes were kept constant for all measurements.To increase the proportion of the Pr state as much as possible, 1 mL of purified phyA proteins was treated with far-red light laser (740 nm, 50.41 µmol•m −2 •s −1 ) irradiation for 20 min on ice before assay.Pr spectra were collected from 500-800 nm while the scan rate was set at maximum to minimize the red or far-red light exposure.Pr-to-Pfr photoconversion was driven with different fluence red light laser (660 nm, 15 µmol for 30 min on ice, and then collected Pfr spectra as above.The Pfr spectrum was subtracted from the Pr spectrum to generate difference spectra.Time-resolved Pr-to-Pfr photoconversion data in Fig. 1m and Fig. S12 were collected in red light irradiation by Cary 4000 UV-Vis Spectrophotometer (Agilent) after driving the Pr levels to steady state with a 740 nm LED.
Pr-to-Pfr photoconversions were driven by LED with peak emissions of 670 nm and the fluence rate of 30 µmol•m −2 •s −1 .Simulated kinetic profiles for photoconversion as monitored by the gain in Pfr absorption at 720 nm to show the relative absorption changes for the samples.

Cryo-EM sample preparation and data collection
For cryo-EM sample preparation, 3.5 μL aliquot of apo-AtphyA, AtphyA-Pr, and ZmphyA1-Pr sample were applied to discharged 300-mesh Au R1.2/1.3 grids (C-flat).Grids were blotted for 2-3.5 s and plunged into liquid ethane using an FEI Mark IV Vitrobot operated at 4 °C and 100% humidity.Cryo-EM data were collected on a Titan Krios (ThermoFisher Scientific) electron microscope operated at 300 kV and a Gatan K3 Summit direct electron detection camera (Gatan) using EPU software.Micrographs were recorded in super-resolution mode at a nominal magnification of 105,000 ×, resulting in a physical pixel size of 0.85 Å per pixel.Defocus values varied from -1 μm to -2 μm.The dose rate was 15 electron per pixel per second.Exposures of 2.41 s were dose-fractionated into 40 subframes, leading to a total accumulated dose of 50 electrons per Å 2 .

Image processing and 3D reconstruction
For the apo-AtphyA, 8,117 movies were collected.Motion correction were performed using MotionCor2 (ref.1) and the contrast transfer function (CTF) parameters were estimated with the Gctf 2 .RELION 3 (versions 4.0) were used for particle picking, 2D and 3D classification and structural refinement.6,300,392 autopicked particles were subjected to 2D classification, resulting in a dataset of 2,601,275 particles for further processing.After rounds of 2D and 3D classification, the particles were divided to three groups and subjected to 3D refinement.To explore the structural dynamics of PhyA, each group particles were subjected to 3D classification.The resulting classes with similar orientation were combined and subjected to further 3D refinement.As the flexibility of HK domain, masked 3D classification and refinement was used to obtain high resolution structure.A total of 873,384 particles were kept for final 3D refinement and model building (final resolution 3.77 Å).Parameters for data processing and 3D reconstruction are summarized in Figure S4 and Table S1.
For AtphyA-Pr, 10,864 movies were collected.The raw super-solution dose-fractionated images stacks were 2 × Fourier binned, aligned, dose-weighted and summed using Patch Motion Correction in cryoSPARC 4 (versions 4.1.2),resulting in summed micrographs in a pixel size of 0.85 Å per pixel.Contrast transfer function (CTF) parameters were estimated using Patch CTF Estimation in cryoSPARC.After excluding the micrographs with bad Thon rings, 10,457 micrographs were selected for subsequent processing.A total of 8,006,050 particles were autopicked using cryoSPARC Template Picker.2D templates used for particles auto-picking was generated by emd_24780.Then several rounds of reference-free 2D classification were performed to remove contaminants and noise in the raw automatically picked particles.The selected particles after 2D classification were subjected to ab-initio reconstruction requesting three classes in cryoSPRC.Then 3D classification was performed using the 3D map of the best class in the last step as the initial reference model.A total of 774,153 particles were selected for the final 3D autorefinement.The AtphyA-Pr yielded a particle density with an estimated resolution of 3.03 Å for model building.Reconstruction resolutions were determined based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion with the high-resolution noise substitution.Parameters for data processing and 3D reconstruction are summarized in Figure S2 and Table S1.
For ZmphyA1-Pr, it was processed similarly to the AtPhyA-Pr dataset, as shown in Figure S3 and Table S1.8,996 CTF-corrected cryo-EM images were manually selected and totally extracted 6,942,083 particles for 2D classification and 3D classification.Finally, 229,726 particles were refined and subjected to 3D refinement at 3.33 Å resolution in an overall structure.

Model building and refinement
The predicted 3D model of AtphyA and ZmphyA1 from Alpha Fold 5 was used as the initial template.The model of AtphyA and ZmphyA1 was built into the map of the dimeric phyA-Pr complex by using Coot 6 .For AtphyA-Pr, model of apo-AtphyA was fitted as rigid bodies followed by manual adjustment and rebuilding in Coot.The PDB file of PΦB was generated from the cryo-EM structure corresponding to PDB file 7rzw.The automatic model refinement was performed by real-space refinement (phenix.real_space_refine) in Phenix 7 and manual adjustment in COOT.

ADDITIONAL DISCUSSION
In this study, we solved cryo-EM structures of apo-AtphyA, AtphyA-Pr and ZmphyA1-Pr, which revealed similar asymmetric homodimers.Structural comparison of apo-AtphyA and AtphyA-Pr showed that PΦB binding induces reorganization of the elements composing the PΦB-binding cradle.The nearly identical structures of ZmphyA1-Pr and AtphyA-Pr agree with their spectral characteristics 9 , further strengthening the conclusion that conserved mechanisms are involved in phyA-mediated light sensing in monocots and dicots.Intriguingly, a putative Class Ⅰ nuclear localization signal (NLS) is included in the 380s loop (residues N344-K361) in phyA-GAF 10 .A chimeric phyB protein with its GAF substituted by phyA-GAF is localized to the nucleus in darkness 11 .Interestingly, replacement of the C-terminal region of the chimaera with the corresponding part of phyA cancels the nuclear localization phenotype.Hence, the NLS in the 380s loop is thought to be sequestered by the C-terminus of phyA.However, the 380s loop in the full-length structure of phyA containing the PAS2 and HKRD domains is flexible (Supplementary information, Fig. S8a), possibly wobbling at the opposite side of the PSM-PAS2 platform relative to HKRDs, making it difficult to explain the sequestration of the NLS by the C-terminal region.A previous study suggested that phyA is an intrinsic kinase with the nPAS-GAF-PHY module required for kinase activity and a putative ATP-binding pocket formed by the α1 and α7 helices of the PHY domain 12 .However, tight packing of two helices of the PHY domain in the putative pocket leaves little room to accommodate an ATP molecule (Supplementary information, Fig. S8e).
Despite their conserved PΦB-binding mechanisms, AtphyA-Pr and AtphyB-Pr differ in their Nterminal PSMs.The PSM of AtphyA-Pr is stretched by a less twisted tongue from the PHY domain, giving rise to a slightly wider PSM-PAS2 platform.Structural alignment showed that PΦB binding induces no striking conformational changes in AtphyA-Pr and that the bound PΦB is completely buried in a phy protein, suggesting that the local conformational changes in the PΦB binding site may be sufficient for signalling.PHY tongue contact with PΦB is essential for Pr to Pfr photoconversion [13][14][15] .A PHY-deleted version of AtphyB generated a normal Pr conformer but could not be photoconverted to the Pfr state 16 , consistent with the role of PHY in stabilizing Pfr conformers.Specific residue mutations in the tongue of the PHY domain altered the Pr/Pfr interconversion and the photosensitivity of phys [16][17][18][19] .In addition, the combination of the PHY tongue with the approaching modulator loop in PAS2 was speculated to compose a photostate sensing interface 20 .Given that the PHY tongue of phys is transformed into a helical structure to stabilize their Pfr conformers, as observed in bacterial relatives 21,22 , the structural divergences in the tongue of phyA-Pr and phyB-Pr are expected to affect the transformation efficiency and consequently their photosensitivities.
When phyA controls physiological processes, it typically mediates VLFR when etiolated seedlings are still under the soil surface and another photoresponse, FR-HIR, in a far-red lightenriched environment, such as under leaf canopies.Hence, phyA is well known as a highly sensitized far-red light photoreceptor.It is also of evolutionary significance for the emergence of phyA in early angiosperms, as the properties of phyA provide adaptive advantages in a shady environment dominated by gymnosperms and ferns.Given that the structure of phyA-Pr offers adequate biological relevance to its high photosensitivity, how can the role of phyA as a far-red light photoreceptor be explained?For both phyA and phyB in the equilibrium of Pr and Pfr interconversion, red light effectively induces most Pfr conformers, and far-red light merely induces traces of Pfr conformers (approximately 2% of the total phy proteins).Hence, a less stable Pr conformer for an easier and quicker transformation of Pr to Pfr and a stronger Pfr conformer to counterbalance the trace amounts seem to be essential for phyA as a far-red light photoreceptor.
The structure of phyA-Pr correlates with the former; however, the structure of its Pfr conformer is urgently needed to address this long-term challenge.

Figure
Figure preparation and structure analysis were performed with PyMOL (pymol.org)and UCSF ChimeraX 8 .

Fig. S1
Fig. S1 Purification and absorbance spectra of phyA proteins.a. Left: Gel filtration profiles of the AtphyA-Pr (black) and apo-AtphyA (red) proteins.Positions of standard molecular weights are indicated by the black arrows.right: Peak fractions in the left were visualized by SDS-PAGE followed by Coomassie blue staining (left) and zincinduced fluorescence assay (right).b.Left: Gel filtration profiles of ZmphyA1-Pr (black) and apo-ZmphyA1 (red) proteins.Positions of standard molecular weights are indicated by black arrows.Right: Peak fractions in the top were visualized by SDS-PAGE followed by Coomassie blue staining (left) and zincinduced fluorescence assay (right).c.Left: UV-vis absorbance spectra of apo-AtphyA (dashed black line), AtphyA proteins in Pr (black line), and after saturating irradiation with red light (red line, Pfr increased).The spectral change ratio (SCR) at 725 nm is 1.17.Right: a cyan color of AtphyA-Pr and the colorless apo-AtphyA.d.Left: UV-vis absorbance spectra collected from apo-ZmphyA1 (dashed black line), ZmphyA1 proteins in Pr (black line), and after saturating irradiation with red light (red line, Pfr increased).Right: a cyan color of ZmphyA1-Pr and the colorless apo-ZmphyA1.

Fig. S2
Fig. S2 Workflow and resolution estimation for the cryo-EM map of AtphyA-Pr.a. Flowchart for data processing of the cryo-EM micrographs of AtphyA-Pr.b.A representative cryo-EM micrograph of AtphyA-Pr.c. Representative views of 2D class averages of the AtphyA-Pr.d.Color-coded local resolution of the final 3D cryo-EM map of the AtphyA-Pr.e. Eulerian angle distribution of raw particle images used in the final 3D refinement.f.Gold-standard Fourier shell correlation (FSC) curve for the final 3D refinement and the validation of the atomic model by correlation curves comparing the model to the final and half map.

Fig. S3
Fig. S3 Workflow and resolution estimation for the cryo-EM map of ZmphyA1-Pr.a. Flowchart for data processing of the cryo-EM micrographs of ZmphyA1-Pr.b.A representative cryo-EM micrograph of ZmphyA1-Pr.c. Representative views of 2D class averages of the ZmphyA1-Pr.d.Color-coded local resolution of the final 3D cryo-EM map of the ZmphyA1-Pr.e. Eulerian angle distribution of raw particle images used in the final 3D refinement.f.Gold-standard Fourier shell correlation (FSC) curve for the final 3D refinement and the validation of the atomic model by correlation curves comparing the model to the final and half map.

Fig. S4
Fig. S4 Workflow and resolution estimation for the cryo-EM map of apo-AtphyA.a. Flowchart for data processing of the cryo-EM micrographs of apo-AtphyA.b.A representative cryo-EM micrograph of apo-AtphyA.c.Representative views of 2D class averages of the apo-AtphyA.d.Color-coded local resolution of the final 3D cryo-EM map of the apo-AtphyA.e. Eulerian angle distribution of raw particle images used in the final 3D refinement.f.Gold-standard Fourier shell correlation (FSC) curve for the final 3D refinement and the validation of the atomic model by correlation curves comparing the model to the final and half map.

Fig. S5
Fig. S5 Overall structures of phyA-related asymmetric dimers.a. Color-coded domain architecture of full-length ZmphyA1.The N-terminal PSM containing NTE, nPAS, GAF and its knot lasso (KL), PHY and its tongue, the internal PAS1-PAS2 along with the modulator loop (Mod), the C-terminal HKRD comprising DHp and CA subdomains are shown.The boundaries are indicated by the numbers.b.Cryo-EM map of ZmphyA1-Pr superposed with atomic model in cartoon shown in three orientations.c.Cryo-EM map of apo-AtphyA superposed with atomic model in cartoon shown in three orientations.d.The low-pass filtered cryo-EM map of AtphyA-Pr is fitted into that of ZmphyA1-Pr to compare the packing of their PAS1s.The fitted model in cartoon is AtphyA-Pr.

Fig. S6
Fig. S6 Topology of AtphyA-Pr generated from its structure model.Topological schematic of AtphyA-Pr.The secondary structural elements of the N-terminal PSM containing NTE, nPAS, GAF and its knot lasso (KL), PHY and tongue, the internal PAS1-PAS2 along with the modulator loop (Mod), the C-terminal HKRD comprising DHp and CA subdomains are shown.Only the α1, α2, α4 helices in CA subdomains are well defined in our cryo-EM structure.

Fig. S7
Fig. S7 Amino acid sequence alignment of phyA and phyB from different species.The positions of the NTE, nPAS, GAF, PHY, PAS1, PAS2, and DHp, CA subdomains of the HKRD are located by the red, blue, green, orange, gray, purple, brown and cyan bars, respectively.The 150s and 380s loops, the knot lasso, the tongue and the modulator loops are located by light blue, light green, dark green, yellow, and dark red bars, respectively.The red box indicates residues linking PHY and PAS1 domains.The solid red circles indicate the residues involved in PΦBbinding.At: Arabidopsis thaliana; Zm: Zea mays; Gm: Glycine max; Os: Oryza sativa.

Fig. S8
Fig. S8 Structure of the N-terminal PSM in phyA-Pr and the PΦB-binding mechanism.a.The PSM of AtphyA-Pr (color-coded) is similar to that of ZmphyA1-Pr (salmon).The helical spine connecting the GAF and PHY domains, the 150s loop in nPAS and 380s loop in GAF are indicated.Residues from D540 to A545 in the PHY tongue are missing in the structure of AtphyA-Pr and residues including the tongue tip cannot be modelled in the structure of ZmphyA1-Pr.b.The PSMs in two protomers of AtphyA-Pr and ZmphyA1-Pr are identical.c.The PSM of AtphyA-Pr (color-coded) is well aligned with the nPAS-GAF fragment of GmphyA-Pr (cyan, PDB code: 6TC7).d.The cyro-EM densities (grey mesh) well define the residues (shown in stick) modelled in the NTE and knot lasso (KL) of GAF domain in AtphyA-Pr.e.The cyro-EM densities (grey mesh) well define the residues (shown in stick) modelled in the tongue (top), α1 and α7 helices (bottom) of PHY domain in AtphyA-Pr.f.Top: The cyro-EM densities (grey mesh) of PΦB (shown in stick) modelled in protomer a (left) and b (middle) of AtphyA-Pr.Shown at right is the alignment of PΦBs in AtphyA-Pr and AtphyB-Pr.Bottom: The cyro-EM densities (grey mesh) define the PΦB (shown in stick) modelled in protomer a (left) and b (right) of ZmphyA1-Pr.g.Top: The PΦB-binding pocket in AtphyA-Pr and the interacting residues from the NTE, GAF and PHY tongue are shown in stick.The dashed yellow lines indicate hydrogen bonds.Bottom:The cyro-EM densities (grey mesh) well define the residues (shown in stick) involved in PΦBbinding shown in top.

Fig. S9
Fig. S9 Comparison of the PΦB-binding pocket in AtphyA-Pr and apo-AtphyA.a.The key residues (shown in stick) involved in PΦB binding in the GAF helix-α5 (left) and the other parts of its pocket (right) of AtphyA-Pr (green) and apo-AtphyA (grey) are superposed.b.The cyro-EM densities (grey mesh) well define the residues (shown in stick) involved in PΦBbinding in the GAF helix-α5 (left) and the other parts of its pocket (right) of apo-AtphyA.

Fig. S10
Fig. S10 Structure of the C-terminal region in phyA-Pr and the dimerization mechanism.a.The PAS2-(nPAS-GAF) interprotomer interface (left) and PAS2-PHY intraprotomer interface (right) in AtphyA-Pr (color-coded) and AtphyB-Pr (grey, PDB code: 7RZW).The helices and strands composing the interfaces, the modulator loop (Mod) in PAS2 and tongue in PHY are indicated.In the PAS2-(nPAS-GAF) interprotomer interface, α3 and α4 of PAS2 contact α1 and α6 of GAF and a small turn in the nPAS of the other protomer primarily through hydrophobic interactions.In the PAS2-PHY intraprotomer interface, PAS2 packs against its adjacent PHY mainly via the interactions between the β5 strand in the PAS2 and the α1 helix in the PHY.b.The cyro-EM densities (grey mesh) well define the residues (shown in stick) modelled in the modulator loop (Mod) of PAS2 domain in AtphyA-Pr.c. Left: Interactions in the PAS2-(nPAS-GAF) interprotomer interface in AtphyA-Pr.The interacting residues are shown in stick.The dashed yellow and red lines indicate hydrogen bonds and electrostatic interactions, respectively.Right: The cyro-EM densities (grey mesh) well define the residues (shown in stick) involved in PAS2-(nPAS-GAF) interprotomer interface shown in left.d.Comparison of critical residues involved in PAS2-(nPAS-GAF) interprotomer interface between AtphyA-Pr and AtphyB-Pr.Dashed lines indicate the conserved amino acids.e.The PSM-PAS2 modules in two protomers of AtphyA-Pr and ZmphyA1-Pr are identical.f.The PAS2-(nPAS-GAF) interprotomer interface and PAS2-PHY intraprotomer interface are conserved in AtphyA-Pr (color-coded) and ZmphyA1-Pr (salmon).g.The cyro-EM densities (colored surface) well define the secondary structure elements (shown in cartoon) containing the H1 and H2 helices of DHp subdomain, and α1, α2, α4 helices in CA subdomains of HKRD in AtphyA-Pr.h.Conserved "head to head" asymmetric homodimers of HKRDs in AtphyA-Pr (color-coded) and ZmphyA1-Pr (salmon).A kink occurs to H1 of protomer b, resulting in the H1a(b) and H1b(b).i. Asymmetric HKRD homodimers of AtphyA-Pr (color-coded) and AtphyB-Pr (grey, PDB code: 7RZW) are shown in four orientations.Both the two H1 helices in AtphyB-Pr possess kinks.The four-helix bundle containing H1b and H2 helices in DHp subdomains and its tightly packing CA subdomains are well superposed in the two phys.The two H1a helices are not

Fig. S11
Fig. S11 Structures of phyA-Pr are conserved in monocot and dicot.a. Structural comparison of AtphyA-Pr (color-coded) and ZmphyA1-Pr (salmon) shown in three orientations.The two structures are well superposed and their tilting angles of HKRD dimers are same.b.Structural comparison of AtphyA-Pr (color-coded) and ZmphyA1-Pr (salmon) shown in another two orientations to highlight no contacts between HKRDs and PSMs in ZmphyA1-Pr.c.Sequence alignment of DHp subdomains in different phys indicates the sequences of kink regions are not conserved.At: Arabidopsis thaliana; Os: Oryza sativa; Zm: Zea mays; Gm: Glycine max.

Fig. S12
Fig. S12 Purification and spectral characteristics of phyA-ΔHKRD proteins.a. Left: Gel filtration profiles of phyA-ΔHKRD proteins.Positions of standard molecular weights are indicated by the black arrows.Right: Peak fractions in the left were visualized by SDS-PAGE followed by Coomassie blue staining (left) and zinc-induced fluorescence assay (right).b.UV-vis absorbance spectra of full-length AtphyA proteins in Pr (black line), after saturating irradiation with red light (red line, Pfr increased).SCR is indicated in parenthesis.Spectra were measured by three technical replicates.c.UV-vis absorbance spectra of HKRD-truncated AtphyA proteins in Pr (black line), after saturating irradiation with red light (red line, Pfr increased).SCR is indicated in parenthesis.Spectra were measured by three technical replicates.d.The normalized UV-vis absorbance spectra shown in b and c.